The broad goal of this research is to understand how nature has adapted the redox-sensing transcription factor SoxR to serve the needs of organisms with different physiologies and environmental challenges. SoxR has traditionally been known as the mediator of an oxidative stress response in Escherichia coli and Salmonella enterica, but this role may be restricted to enteric bacteria. In the vast majority of non-enterics, SoxR is predicted to mediate a response to endogenously-produced redox-active antibiotics. This has been confirmed in Pseudomonas aeruginosa where SoxR senses endogenous antibiotics and controls the expression of genes involved in their transport and processing. The proposed research program is designed to examine the function and redox-sensing properties of SoxR in the evolutionarily divergent antibiotic producer Streptomyces coelicolor. S. coelicolor produces several secondary metabolites including two redox-active pigmented antibiotics, actinorhodin and undecylprodigiosin. Deletion of soxR in S. coelicolor causes accelerated development and hyper-production of these pigmented antibiotics, suggesting that SoxR participates in a regulatory network that controls developmental and antibiotic-metabolizing genes. The expression of two SoxR-target genes is significantly reduced in a pigment-deficient mutant suggesting that, as in P. aeruginosa, SoxR may mediate its effects in response to endogenous antibiotics in S. coelicolor. Microarray studies will be conducted to identify genes that are directly and indirectly regulated by SoxR as S. coelicolor proceeds through its developmental cycle. These will be independently verified by quantitative real time PCR and direct SoxR-targets will be confirmed by gel shift assays in vitro. The identification of the SoxR regulon will help to forge a better understanding of how SoxR regulates morphological and physiological differentiation in this model antibiotic-producer. The mechanism of redox-sensing by S. coelicolor SoxR will be investigated in vivo. The importance of the [2Fe-2S] clusters will be assessed by examining the ability of a cluster-deficient mutant to complement the defects in the ?soxR mutant. The same strategy will be employed to probe the importance of a potential regulatory domain consisting of an extended C-terminal region (with two cysteine residues) that is unique to SoxRs from Streptomyces species. The transcriptional activity of SoxR in an actinorhodin-deficient mutant, and separately in an undecylprodigiosin-deficient mutant will be examined to identify the relevant physiological signal. Finally, the ability of exogenously added redox-cycling chemicals to elicit SoxR activity will be assessed in a pigment-deficient mutant, which will reveal specificity of signal recognition. Together, these experiments will provide further insight into redox-sensing and transduction mechanisms by iron-sulfur regulatory proteins. Finally, as an AREA proposal, the research will train undergraduate students in the design and execution of hypothesis-driven experimentation and data analysis. PUBLIC HEALTH RELEVANCE: Members of the Streptomyces genus are notable for producing two-thirds of the biologically active metabolites (including antibiotics) used in clinical and veterinary medicine. The indication that SoxR controls cellular machinery to process and/transport antibiotics in the model S. coelicolor, has implications for optimizing the production of bioactive molecules by Streptomyces, and may further bring new candidates to our dwindling list of effective antibiotics.